(A) Size profile of DNA fragments from CpG island library. 3H9R/Vκ8R B cells were stimulated with ssDNA (B) in the presence or absence of the TLR9-inhibitor S-ODN 2088 (C). AM14 B cells were stimulated with dsDNA fragment ICs (5 μg/ml Hy1.2 + TNP-dsDNA) (D) or with dsDNA fragment ICs (5 μg/ml Hy1.2 + 10 ng/ml TNP-dsDNA) containing mouse genomic DNA, PCR-amplified unmethylated mouse genomic DNA (average length 400 bp), or PCR-amplified CpG island DNA (E). WT B6 or anti-HEL/RAG^−/− B cells were stimulated with dsDNA fragments (F). DNA was from E coli ■, CG50 △, CpG islands ○, mouse genome ▲, or CGneg □. Proliferation was assessed by ³H-thymidine incorporation and results are standardized as percent proliferation induced by PS-ODN 1826. The data shown are the mean ± SEM of 3 experiments (B, D) 4 experiments (E), or are representative of 2 experiments (C, F).

(A) 3H9R/Vκ8R B cells were stimulated with ssDNA (300 ng/ml). (B) AM14 B cells were stimulated with fragment ICs (5 μg/ml Hy1.2 or 1D4 + 100 ng/ml TNP-dsDNA or bio-dsDNA). Results are presented as percent proliferation relative to either clone 5 (A) or PS-ODN 1826 (B) and are the mean ± SEM of 3 experiments. (C, D) Increasing concentrations of unlabeled ssDNA were added to plate-bound 3H9R/Vκ8R IgM along with biotinylated CG50 fragment. Results are representative of 3 experiments.

(A) Samples containing a fixed amount of clone 11 dsDNA prepared with increasing percentage of biotinylated bases were incubated alone in the presence or absence of the 1D4 anti-biotin mAb and then electrophoresed on a 1% agarose gel. Similar results were obtained with CGneg and Senp1 (data not shown). (B) The same DNA-bio/1D4 ICs were added to AM14 B cells for 1 hr at 4°C and level of IC bound to the cell was determined by flow cytometry by staining with goat anti-mouse IgG2a-FITC. Results are representative of 2 experiments. (C) AM14R B cells were stimulated with 15 μg/ml F(ab')₂ goat anti-mouse IgM, 1 μg/ml PS-ODN 1826, 5 μg/ml PA4, or dsDNA ICs (5 μg/ml 1D4 + 100 ng/ml biotinylated dsDNA) for 10 min and cell lysates were assayed for phospho-tyrosine by western blot. β-actin is shown as a loading control. (D) AM14R B cells were stimulated as above and total cell lysates were immunoprecipitated with anti-Syk followed by western blot with anti-phosphotyrosine. (E) AM14R B cells were stimulated with 5 μg/ml 1D4, or fragment ICs (5 μg/ml 1D4 + 100 ng/ml biotinylated dsDNA) and Ca²⁺ flux was measured by flow cytometry. (F) AM14R B cells were stimulated with ICs (5 μg/ml 1D4 + 100 ng/ml biotinylated dsDNA) and proliferation was measured. Results are representative of 3 experiments (C, D), 2 experiments (E), or are the mean ± SEM of 3 experiments (F).

Proliferation of AM14 (A) and AM14/TLR9^−/− (B) B cells in response to 15 μg/ml F(ab')₂ goat anti-mouse IgM, 100 ng/ml CLO97, 1 μg/ml PS-ODN 1826, or fragment ICs (5 μg/ml 1D4 + 100 ng/ml of biotinylated dsDNA) in the presence or absence of 1000 U/ml IFN-α. (C, D) Proliferation of AM14R B cells in response to 5 μg/ml 1D4, 1 μg/ml OVA-bio, protein IC (5 μg/ml 1D4 + 300 ng/ml OVA-bio), or DNA ICs (5 μg/ml 1D4 + 100 ng/ml biotinylated mouse dsDNA or clone 11) in the presence or absence of 1000 U/ml IFN-α. Results are the mean ± SEM of 3 experiments. (E) Proliferation of AM14R B cells in response to increasing concentrations of PS-ODN 1826 or fragment ICs (1.5 μg/ml 1D4 + biotinylated dsDNA) in the presence or absence of 1000 U/ml IFN-α; results are representative of 3 experiments. AM14R B cells were incubated for 24 hrs in the presence or absence of 1000 U/ml IFN-α and surface levels of CD69 and IgM were measured by flow cytometry (F), or they were stimulated with fragment ICs (1.5 μg/ml 1D4 + 30 ng/ml biotinylated dsDNA), 5 μg/ml F(ab')₂ goat anti-mouse IgM, or 1 μg/ml PS-ODN 1826 and Ca²⁺ flux was measured by flow cytometry (G), or cell lysates were assayed for phospho-PLCγ2 (H); results are representative of 2 experiments.